IL-17 (or CTLA-8), a cytokine secreted by Th17 cells and the like, is profoundly associated with inflammatory diseases, autoimmune diseases, and infectious diseases. Human IL-17 is a 20-30 kDa glycoprotein, constituted of 155 amino acids, comprising a signal peptide at the N-terminus. In the molecular structure thereof, six cysteine residues and one N-binding sugar chain binding site are present. The mature form consists of 136 amino acids, normally occurring as a dimer.
As proteins of the IL-17 family, six kinds of proteins are known: IL-17A, B, C, D, E, and F. Generally, IL-17 refers IL-17A. IL-17E is also called IL-25. The amino acid sequence homology of human IL-17 to human IL-17B, C, D, E, and F is 25, 28, 22, 27, and 44%, respectively, IL-17F being of the highest homology. Human IL-17 has a homology of 63% to mouse IL-17. As receptors thereof, IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE are known. IL-17 and IL-17F form a homodimer or heterodimer and bind to IL-17RA and IL-17RC. The binding of IL-17 and IL-17RA is weak at a Kd value of about 10−7, and involvement of IL-17RC may be important.
The Th17 cells are CD4+ T cells that produce IL-17. When Th17 cells are stimulated with IL-23 in vitro, IL-17 production is induced. Meanwhile, TGF-β and IL-6 play an important role in the differentiation induction of Th17 cells. TGF-β and IL-6 act on naïve T cells to induce the expression of RORγt (transcription factor). Because a deficiency in RORγt prevents Th17 cells from being differentiated, and also because naïve T cells can conversely be differentiated into IL-17-producing cells by forcedly expressing RORγt, this transcription factor is thought to be important to the differentiation of Th17 cells. Although activation of STAT3 by IL-6 is important to the induction of the expression of RORγt, activation of STAT5 by IL-2 conversely suppresses the expression. IL-2 is necessary for the differentiation of regulatory T cells; IL-2-deficient mice show serious autoimmunity; this is thought to be due to a decrease in regulatory T cells along with over-differentiation of Th17 cells. When naïve T cells are stimulated with TGF-β alone in vitro, not Th17, but regulatory T cells, are induced. IFN-γ produced by Th1 cells, IL-4 produced by Th2 cells, and the like act suppressively on the differentiation of Th17 cells.
When IL-17 binds to an IL-17 receptor, the NF-κB pathway, MAP kinase pathway, and C/EBP pathway are activated via Act-1 and TRAF6, resulting in the induction of inflammatory cytokines and chemokines. For example, IL-17 acts on macrophages to induce the expression of IL-1, TNF and the like. In addition, IL-17 is known to act also on connective tissue cells and epithelial tissue cells such as fibroblasts and endothelial cells, and on immune system cells such as dendritic cell progenitors, to induce the expression of various receptors and cytokines such as IL-6 and IL-1.
Cytokines such as TNF-α, IL-1β, and IL-6 are involved in the production of IL-17. Meanwhile, production of these cytokines is induced by IL-17. IL-17 is known to act synergistically with other cytokines.
It has been found that IL-17 is profoundly associated with inflammatory diseases, autoimmune diseases and the like. It is known that the expression of IL-17 is elevated in patients with rheumatoid arthritis, age-related macular degeneration, psoriasis, systemic lupus erythematosus, Behçet's disease, graft rejection, nephritic syndrome, inflammatory bowel disease, asthma, multiple sclerosis, periodontal disease and the like. In IL-17-deficient mice, it has been reported that collagen-induced arthritis (CIA), which is a model of rheumatoid arthritis; experimental autoimmune encephalomyelitis (EAE), which is a model of multiple sclerosis; contact type hypersensitivity reactions by DNFB or TNCB; delayed type hypersensitivity reactions by methylated BSA; airway hypersensitive reactions by OVA induction, and the like are remarkably suppressed.
IL-17 is also associated with cancers. It has been reported that subcutaneous transplantation of non-small cell lung cancer cells to SCID mice promotes the proliferation of cancer cells in mice having IL-17 expressed highly therein. It has also been reported that IL-17 is also associated with cervical cancer and ovarian cancer.
IL-17 is associated with infectious diseases. IL-17 receptor knockout mice are highly susceptible to Klebsiella pneumoniae infection, Candida albicans infection, Toxsoplasma gondii infection and the like. IL-17 production is induced by lipopolysaccharides (LPS) and bacterial cell body components such as of Borrelia burgdorferi and Klebsiella pneumoniae. These components are thought to promote IL-17 production by acting on antigen-presenting cells to induce IL-23. In IL-17R-knockout mice, after Klebsiella pneumoniae infection, in infected sites in the lung, the production of CXCL1, CXCL2, G-CSF and the like, which play an important role in the migration and functions of neutrophils, is reduced and the migration of neutrophils is suppressed.
In recent years, applications of RNA aptamers to therapeutic drugs, diagnostic reagents, and test reagents have been drawing attention; some RNA aptamers have already been in clinical study stage or in practical use. In December 2004, the world's first RNA aptamer drug, Macugen, was approved as a therapeutic drug for age-related macular degeneration in the US. An RNA aptamer refers to an RNA that binds specifically to a target molecule such as a protein, and can be prepared using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method (cf. Patent document 1, 2, and 3). In the SELEX method, an RNA that binds specifically to a target molecule is selected from an RNA pool with about 1014 different nucleotide sequences. The RNA used has a random sequence of about 40 residues, which is flanked by primer sequences. This RNA pool is allowed to mix with a target molecule, and only the RNA that has bound to the target molecule is collected using a filter and the like. The RNA collected is amplified by RT-PCR, and this is used as a template for the next round. By repeating this operation about 10 times, an RNA aptamer that binds specifically to the target molecule can be obtained.
Aptamer drugs, like antibody drugs, can target extracellular factors. With reference to many scientific papers and other reference materials in the public domain, there is a possibility that aptamer drugs surpass antibody drugs in some aspects. For example, aptamers often show higher binding force and higher specificity than antibodies do. Aptamers are unlikely to undergo immune elimination, and adverse reactions which are characteristic of antibodies and result from antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), do not occur with the use of aptamers. From the aspect of delivery, since aptamers are about 1/10 of antibody in size, delivery of a drug to the object site is easier. Since aptamers are produced by chemical synthesis, various modifications can be done easily, and reduction of cost by large-scale production is possible. Meanwhile, the blood half-lives of aptamers are generally shorter than those of antibodies; however, this property is sometimes advantageous in view of toxicity. These facts lead to the conclusion that even when the same molecule is targeted, aptamer drugs potentially surpass antibody drugs.
Patent document 4 describes an aptamer obtained by the above-mentioned SELEX method, which binds to IL-17 to inhibit binding of IL-17 and IL-17 receptor.